A family of molecules is referred to as the "TGF-.beta.s". These are 25 kd dimeric proteins which have multi-functional effects on growth and differentiation of cells, both in vitro and in vivo. See Roberts et al. in Peptide Growth Factors And Their Receptors I (Sporn et al., eds., pp 419-472; Springer-Verlag, Berlin, 1990); Moses et al., Cell 63: 245-247 (1990); Massague, Ann. Rev. Cell. Biol. 6: 597-641 (1990). The family contains at least three different, structurally related members, identified as ".beta.1, .beta.2 and .beta.3". Many other proteins are more distantly related, including bone morphogenic proteins, Mullerian inhibitory substance, activins, inhibins, and so forth.
Originally, the TGF-.beta. family of proteins was identified as being involved in increasing anchorage independent growth of normal rat kidney cells; however, the proteins are also recognized as a potent growth inhibitor for diverse cell types, including hematopoietic cells, lymphocytes, epithelial and endothelial cells (Ohta et al., Nature 329: 539-541 (1987); Kehri et al., J. Immunol 137: 3855-3860 (1986); Moses et al., in Cancer Cells 3 (Feramisco et al., ed; Cold Spring Harbor, N.Y., 1985); pg. 65-71; Baird et al.. Biochem. Biophys. Res. Commun 138: 476-482 (1986); Frater-Schroder et al., Biochem. Biophys. Res. Commun. 137: 295-302 (1986); Heimark et al., Science 233: 1078-1080 (1986)). The molecules have a dramatic effect on accumulation of extracellular matrix proteins (Massague, supra), and have been implicated in pathogenesis glomerulonephritis (Border et al., Nature 346: 371-374 (1990)); liver cirrhosis (Castilia et al., N. Eng. J. Med. 324: 933-940 (1990)); and pulmonary fibrosis (Khalil et al., in Clinical Application of TGF-.beta.1 (Bocket al., ed. Ciba Foundation Symposium 157, John Willy & Sons, 1991, pg. 194-211).
The TGF-.beta. family interacts with other proteins on several levels. One of these is mediation of binding via cell surface receptors. The art recognizes three distinct high affinity receptors for TGF-.beta.s, referred to as types I, II and III. The first two of these have molecular masses of 53 and 70-85 kd, respectively, while the third is denoted "betaglycan" because of its proteoglycan like structure, and is further characterized by a molecular mass of 200-400 kd. Massague et al., in Transforming Growth Factor-.beta.s: Chemistry, Biology and Therapeutics (Piez et al., eds., Ann. N.Y. Acad. Sci. 593, 1990), pg. 59-72; Segarini et al., in Clinical Applications of TGF-.beta. (Bock et al., eds. Ciba Foundation Symposium 157, John Wiley & Sons, 1991, pg 29-50). The betaglycan molecule is a membrane proteoglycan, having a 100-140 kd core protein with unknown functional importance, while type I and II receptors appear to be involved in transduction of TGF-.beta. cellular effect. Segarini et al., J. Biol. Chem. 263: 8366-8370 (1988); Cheifetz et al., J. Biol. Chem. 263: 16884-16991 (1988); Massague et al., supra. Some cell lines express only type I receptors and are inhibited by TGF-.beta.1. These include hematopoietic progenitor cell lines (Ohta et al., supra) and squamous cancer cell lines (Ichiyo et al., Exp. Cell Res. 187: 263-269 (1990)). Mutant cell lines of mink epithelial cells have been shown to have lost or to have anomalous expression of type I and/or type II receptors (Boyd et al., J. Biol. Chem. 264: 2272-2278 (1989); Laiho et al., J. Biol. Chem. 265: 18518-18524 (1990)).
Additional binding molecules for TGF-.beta. having molecular masses of 60 kd, 85-320 kd, and 400 kd have been reported in pituitary tumor cell lines, rat glomeruli, and bovine liver cells, respectively, as reported by Cheifetz et al., J. Biol. Chem. 263: 17225-17228 (1988); Mackay et al., J. Biol. Chem. 265: 9351-9356 (1990); O'Grady et al., J. Biol. Chem. 266: 8583-8589 (1991).
On another level, the precursors of TGF-.beta., especially TGF-.beta.1, interact with protein molecules known as the latent TGF-binding protein or "LTBP". The interaction yields a high molecular weight, inactive complex which is secreted from the cell. This is sometimes referred to as the latent TGF-.beta.1 complex. See Miyazono et al., J. Biol. Chem. 263: 6407-6415 (1988); Pircher et al., Biochem. Biophys. Res. Commun. 136: 30-37 (1984); Wakefield et al., J. Cell Biol. 105: 965-975 (1987). The inactive or latent complexes contain a non-covalent association of TGF-.beta.1, a disulphide bonded complex of a dimer of N-terminal peptide of TGF-.beta.1 precursor and as third component, the LTBP. This third component occurs as a molecule with a molecular mass which may range from 125-190 kds. Experiments have shown that the binding proteins do not inactivate TGF-.beta.1.
The molecules discussed supra are sometimes referred to as "binding proteins", because they do, in fact bind to the TGF-.beta.1 precursor. A fundamental difference between these molecules and the molecules of the invention is that while the prior art molecules may be referred to as "synthesis" binders, it is more appropriate to describe the invention as involving "effector" binders. The synthesis binders are involved in the "packaging" of TGF-.beta.1 in the cell, such that it is released for subsequent activities. When bound to the prior art molecules, TGFs are essentially inert. In contrast, the protein containing molecules of the invention may be seen as "effectors" in that TGF-.beta.1 binds directly to these, so as to effect a response thereby. This distinction should be kept in mind in connection with this application.
It is an object of the invention to describe these substantially pure, receptor like TGF-.beta.1 binding protein containing molecules, which are characterized by molecular masses of 160 kd, 70-80 kd, and 35-40 kd as determined by SDS-PAGE, as well as their uses in various processes. The ranges are due to the behavior of the species under reducing and non-reducing conditions, as will be seen infra. It is also due to the nature of the molecule's structure, which is as a monomer, dimer or trimer based upon a single peptide. The implication of this structure are discussed infra.
The objects of the invention discussed supra as well as others will be seen from the disclosure which follows.